Publications by authors named "Djamel Lebeche"

58 Publications

Direct Reprogramming Induces Vascular Regeneration Post Muscle Ischemic Injury.

Mol Ther 2021 Jul 28. Epub 2021 Jul 28.

Cardiovascular Research Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 10029; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 10029; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 10029. Electronic address:

Reprogramming non-cardiomyocytes (non-CMs) into cardiomyocyte (CM)-like cells is a promising strategy for cardiac regeneration in conditions such as ischemic heart disease. Here, we used a modified mRNA (modRNA) gene delivery platform to deliver a cocktail of four cardiac-reprogramming genes (Gata4 (G), Mef2c (M), Tbx5 (T) and Hand2 (H)) together with three reprogramming-helper genes (Dominant Negative (DN)-TGFβ, DN-Wnt8a and Acid ceramidase (AC)), termed 7G-modRNA, to induce CM-like cells. We showed that 7G-modRNA reprogrammed 57% of CM-like cells in vitro. Through a lineage-tracing model, we determined that delivering the 7G-modRNA cocktail at the time of myocardial infarction reprogrammed ∼25% of CM-like cells in the scar area and significantly improved cardiac function, scar size, long-term survival and capillary density. Mechanistically, we determined that while 7G-modRNA cannot create de-novo beating CMs in vitro or in vivo, it can significantly upregulate pro-angiogenic mesenchymal stromal cells markers and transcription factors. We also demonstrated that our 7G-modRNA cocktail leads to neovascularization in ischemic-limb injury, indicating CM-like cells importance in other organs besides the heart. modRNA is currently being used around the globe for vaccination against COVID-19, and this study proves this is a safe, highly efficient gene delivery approach with therapeutic potential to treat ischemic diseases.
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http://dx.doi.org/10.1016/j.ymthe.2021.07.014DOI Listing
July 2021

Resistin deletion protects against heart failure injury by targeting DNA damage response.

Cardiovasc Res 2021 Jul 29. Epub 2021 Jul 29.

Cardiovascular Research Institute.

Aims: Increased Resistin (Retn) levels are associated with development of cardiovascular diseases. However, the role of Retn in heart failure (HF) is still unclear. Here we probed the functional and molecular mechanism underlying the beneficial effect of Retn deletion in HF.

Methods And Results: Wildtype (WT) and adipose tissue-specific Retn-knockout (RKO) mice were subjected to transverse aortic constriction (TAC) induced HF. Cardiac function and hemodynamic changes were measured by echocardiography and left ventricular catheterization. Adipose tissue Retn deletion attenuated while Retn cardiac-selective overexpression, via a recombinant adeno-associated virus-9 vector, exacerbated TAC-induced hypertrophy, cardiac dysfunction and myocardial fibrosis in WT and RKO mice. Mechanistically, we showed that Gadd45α was significantly increased in RKO HF mice while cardiac overexpression of Retn led to its down-regulation. miR148b-3p directly targets Gadd45α and inhibits its expression. Retn overexpression upregulated miR148b-3p expression and triggered DNA damage response (DDR) in RKO-HF mice. Inhibition of miR148b-3p in vivo normalized Gadd45α expression, decreased DDR and reversed cardiac dysfunction and fibrosis. In vitro Retn overexpression in adult mouse cardiomyocytes activated miR148b-3p and reduced Gadd45α expression. Gadd45α overexpression in H9C2-cardiomyoblasts protected against hydrogen peroxide- and Retn-induced DDR.

Conclusion: These findings reveal that diminution in circulating Retn reduced myocardial fibrosis and apoptosis, and improved heart function in a mouse model of HF, at least in part, through attenuation of miR148b-3p and DDR. The results of this study indicate that controlling Retn levels may provide a potential therapeutic approach for treating pressure overload-induced HF.
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http://dx.doi.org/10.1093/cvr/cvab234DOI Listing
July 2021

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro.

J Vis Exp 2021 05 10(171). Epub 2021 May 10.

Cardiovascular Research Center, The Icahn School of Medicine at Mount Sinai; Diabetes, Obesity and Metabolism Institute, Department of Medicine, The Icahn School of Medicine at Mount Sinai; Graduate School of Biomedical Sciences, The Icahn School of Medicine at Mount Sinai.

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.
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http://dx.doi.org/10.3791/62419DOI Listing
May 2021

Therapeutic Delivery of Pip4k2c-Modified mRNA Attenuates Cardiac Hypertrophy and Fibrosis in the Failing Heart.

Adv Sci (Weinh) 2021 05 12;8(10):2004661. Epub 2021 Mar 12.

Cardiovascular Research Center Icahn School of Medicine at Mount Sinai New York NY 10029 USA.

Heart failure (HF) remains a major cause of morbidity and mortality worldwide. One of the risk factors for HF is cardiac hypertrophy (CH), which is frequently accompanied by cardiac fibrosis (CF). CH and CF are controlled by master regulators mTORC1 and TGF-, respectively. Type-2-phosphatidylinositol-5-phosphate-4-kinase-gamma (Pip4k2c) is a known mTORC1 regulator. It is shown that Pip4k2c is significantly downregulated in the hearts of CH and HF patients as compared to non-injured hearts. The role of Pip4k2c in the heart during development and disease is unknown. It is shown that deleting Pip4k2c does not affect normal embryonic cardiac development; however, three weeks after TAC, adult Pip4k2c mice has higher rates of CH, CF, and sudden death than wild-type mice. In a gain-of-function study using a TAC mouse model, Pip4k2c is transiently upregulated using a modified mRNA (modRNA) gene delivery platform, which significantly improve heart function, reverse CH and CF, and lead to increased survival. Mechanistically, it is shown that Pip4k2c inhibits TGF1 via its N-terminal motif, Pip5k1, phospho-AKT 1/2/3, and phospho-Smad3. In sum, loss-and-gain-of-function studies in a TAC mouse model are used to identify Pip4k2c as a potential therapeutic target for CF, CH, and HF, for which modRNA is a highly translatable gene therapy approach.
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http://dx.doi.org/10.1002/advs.202004661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8132051PMC
May 2021

Leveraging clinical epigenetics in heart failure with preserved ejection fraction: a call for individualized therapies.

Eur Heart J 2021 05;42(20):1940-1958

Center for Molecular Cardiology, University of Zürich, Wagistrasse 12, Schlieren CH-8952, Switzerland.

Described as the 'single largest unmet need in cardiovascular medicine', heart failure with preserved ejection fraction (HFpEF) remains an untreatable disease currently representing 65% of new heart failure diagnoses. HFpEF is more frequent among women and associates with a poor prognosis and unsustainable healthcare costs. Moreover, the variability in HFpEF phenotypes amplifies complexity and difficulties in the approach. In this perspective, unveiling novel molecular targets is imperative. Epigenetic modifications-defined as changes of DNA, histones, and non-coding RNAs (ncRNAs)-represent a molecular framework through which the environment modulates gene expression. Epigenetic signals acquired over the lifetime lead to chromatin remodelling and affect transcriptional programmes underlying oxidative stress, inflammation, dysmetabolism, and maladaptive left ventricular remodelling, all conditions predisposing to HFpEF. The strong involvement of epigenetic signalling in this setting makes the epigenetic information relevant for diagnostic and therapeutic purposes in patients with HFpEF. The recent advances in high-throughput sequencing, computational epigenetics, and machine learning have enabled the identification of reliable epigenetic biomarkers in cardiovascular patients. Contrary to genetic tools, epigenetic biomarkers mirror the contribution of environmental cues and lifestyle changes and their reversible nature offers a promising opportunity to monitor disease states. The growing understanding of chromatin and ncRNAs biology has led to the development of several Food and Drug Administration approved 'epidrugs' (chromatin modifiers, mimics, anti-miRs) able to prevent transcriptional alterations underpinning left ventricular remodelling and HFpEF. In the present review, we discuss the importance of clinical epigenetics as a new tool to be employed for a personalized management of HFpEF.
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http://dx.doi.org/10.1093/eurheartj/ehab197DOI Listing
May 2021

Xanthone glucoside 2-β-D-glucopyranosyl-1,3,6,7-tetrahydroxy-9H-xanthen-9-one binds to the ATP-binding pocket of glycogen synthase kinase 3β and inhibits its activity: implications in prostate cancer and associated cardiovascular disease risk.

J Biomol Struct Dyn 2021 Mar 26:1-17. Epub 2021 Mar 26.

Department of Biochemistry, School of Chemical and Life Sciences, Jamia Hamdard (Deemed University), Delhi, India.

Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase which in the presence of ATP in its ATP-binding pocket transfers a phosphate to a primed substrate. GSK3β is an isoform of GSK3 which has been projected as a potent therapeutic target in human diseases including cancers and metabolic syndrome. Incidentally, cardiovascular disease is a common cause of non-cancer related deaths in prostate cancer (PCa) patients, mainly due to the effects of androgen-deprivation therapy (ADT), a mainstay for PCa treatment. Several small molecular inhibitors of GSK3 are either ATP-competitive (bind to the ATP-binding pocket), or non-ATP-competitive inhibitors (binding to the substrate-binding site of the enzyme). In this study, 2-β-D-glucopyranosyl-1,3,6,7-tetrahydroxy-9H-xanthen-9-one (βDGT), a natural xanthonoid present in many plant species, is reported to bind to the ATP-binding pocket of GSK3β and inhibit its activity, as demonstrated by the molecular docking and molecular dynamics simulation analysis and experimental validation . A comparison of the binding affinities with five known ATP-competitive inhibitors of GSK3β suggested similarity in binding site residues in the ATP-binding pocket of the enzyme. The optimum inhibitory concentration of the xanthonoid as determined by the luminescent kinase assay was 200 µM. The study envisages the use of βDGT as a natural ATP-competitive inhibitor of GSK3β and implicates its use in PCa patients on ADT, a cardiovascular disease risk, and other pathological conditions where GSK3 inhibition may be clinically important. HighlightsGSK3β is a multifaceted kinase known for its role in cancers, cardiovascular, and other diseases.In this study, βDGT, a xanthonoid, is reported to bind to the ATP-binding pocket of GSK3β.A comparison of βDGT binding with 5 known ATP-competitive inhibitors of GSK3β suggested the involvement of residues at the ATP binding site.The binding site analysis suggested an ATP-competitive mechanism of enzyme inhibition.Study envisages the use of βDGT as a natural ATP-competitive inhibitor of GSK3β and implicates its use in prostate cancer patients on androgen-deprivation therapy, a cardiovascular disease risk, and other pathological conditions.Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2021.1902857DOI Listing
March 2021

Proteomic Architecture of Valvular Extracellular Matrix: FNDC1 and MXRA5 Are New Biomarkers of Aortic Stenosis.

JACC Basic Transl Sci 2021 Jan 13;6(1):25-39. Epub 2021 Jan 13.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.

This study analyzed the expression of extracellular matrix (ECM) proteins during aortic valve calcification with mass spectrometry, and further validated in an independent human cohort using RNAseq data. The study reveals that valve calcification is associated with significant disruption in ECM and metabolic pathways, and highlights a strong connection between metabolic markers and ECM remodeling. It also identifies FNDC1 and MXRA5 as novel ECM biomarkers in calcified valves, electing them as potential targets in the development and progression of aortic stenosis.
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http://dx.doi.org/10.1016/j.jacbts.2020.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7838057PMC
January 2021

Resistin induces cardiac fibroblast-myofibroblast differentiation through JAK/STAT3 and JNK/c-Jun signaling.

Pharmacol Res 2021 05 29;167:105414. Epub 2021 Jan 29.

Cardiovascular Research Institute, The Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA; Diabetes, Obesity and Metabolism Institute, Department of Medicine, The Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA; Graduate School of Biomedical Sciences, The Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. Electronic address:

Cardiac fibrosis is characterized by excessive deposition of extracellular matrix proteins and myofibroblast differentiation. Our previous findings have implicated resistin in cardiac fibrosis; however, the molecular mechanisms underlying this process are still unclear. Here we investigated the role of resistin in fibroblast-to-myofibroblast differentiation and elucidated the pathways involved in this process. Fibroblast-to-myofibroblast transdifferentiation was induced with resistin or TGFβ1 in NIH-3T3 and adult cardiac fibroblasts. mRNA and protein expression of fibrotic markers were analyzed by qPCR and immunoblotting. Resistin-knockout mice, challenged with a high-fat diet (HFD) for 20 weeks to stimulate cardiac impairment, were analyzed for cardiac function and fibrosis using histologic and molecular methods. Cardiac fibroblasts stimulated with resistin displayed increased fibroblast-to-myofibroblast conversion, with increased levels of αSma, col1a1, Fn, Ccn2 and Mmp9, with remarkable differences in the actin network appearance. Mechanistically, resistin promotes fibroblast-to-myofibroblast transdifferentiation and fibrogenesis via JAK2/STAT3 and JNK/c-Jun signaling pathways, independent of TGFβ1. Resistin-null mice challenged with HFD showed an improvement in cardiac function and a decrease in tissue fibrosis and reduced mRNA levels of fibrogenic markers. These findings are the first to delineate the role of resistin in the process of cardiac fibroblast-to-myofibroblast differentiation via JAK/STAT3 and JNK/c-Jun pathways, potentially leading to stimulation of cardiac fibrosis.
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http://dx.doi.org/10.1016/j.phrs.2020.105414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8085100PMC
May 2021

Adiponectin receptor 1 variants contribute to hypertrophic cardiomyopathy that can be reversed by rapamycin.

Sci Adv 2021 Jan 6;7(2). Epub 2021 Jan 6.

Cardiovascular Research Center, Department of Medicine, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA.

Hypertrophic cardiomyopathy (HCM) is a heterogeneous genetic heart muscle disease characterized by hypertrophy with preserved or increased ejection fraction in the absence of secondary causes. However, recent studies have demonstrated that a substantial proportion of individuals with HCM also have comorbid diabetes mellitus (~10%). Whether genetic variants may contribute a combined phenotype of HCM and diabetes mellitus is not known. Here, using next-generation sequencing methods, we identified novel and ultrarare variants in adiponectin receptor 1 () as risk factors for HCM. Biochemical studies showed that variants dysregulate glucose and lipid metabolism and cause cardiac hypertrophy through the p38/mammalian target of rapamycin and/or extracellular signal-regulated kinase pathways. A transgenic mouse model expressing an variant displayed cardiomyopathy that recapitulated the cellular findings, and these features were rescued by rapamycin. Our results provide the first evidence that variants can cause HCM and provide new insights into regulation.
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http://dx.doi.org/10.1126/sciadv.abb3991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7787482PMC
January 2021

Boron improves cardiac contractility and fibrotic remodeling following myocardial infarction injury.

Sci Rep 2020 10 13;10(1):17138. Epub 2020 Oct 13.

Cardiovascular Research Centre, The Icahn School of Medicine At Mount Sinai, New York, NY, 10029, USA.

Myocardial fibrosis is a major determinant of clinical outcomes in heart failure (HF) patients. It is characterized by the emergence of myofibroblasts and early activation of pro-fibrotic signaling pathways before adverse ventricular remodeling and progression of HF. Boron has been reported in recent years to augment the innate immune system and cell proliferation, which play an important role in the repair and regeneration of the injured tissue. Currently, the effect of boron on cardiac contractility and remodeling is unknown. In this study, we investigated, for the first time, the effect of boron supplementation on cardiac function, myocardial fibrosis, apoptosis and regeneration in a rat model myocardial infarction (MI)-induced HF. MI was induced in animals and borax, a sodium salt of boron, was administered for 7 days, p.o., 21 days post-injury at a dose level of 4 mg/kg body weight. Transthoracic echocardiographic analysis showed a significant improvement in systolic and diastolic functions with boron treatment compared to saline control. In addition, boron administration showed a marked reduction in myocardial fibrosis and apoptosis in the injured hearts, highlighting a protective effect of boron in the ischemic heart. Interestingly, we observed a tenfold increase of nuclei in thin myocardial sections stained positive for the cell cycle marker Ki67 in the MI boron-treated rats compared to saline, indicative of increased cardiomyocyte cell cycle activity in MI hearts, highlighting its potential role in regeneration post-injury. We similarly observed increased Ki67 and BrdU staining in cultured fresh neonatal rat ventricular cardiomyocytes. Collectively, the results show that boron positively impacted MI-induced HF and attenuated cardiac fibrosis and apoptosis, two prominent features of HF. Importantly, boron has the potential to induce cardiomyocyte cell cycle entry and potentially cardiac tissue regeneration after injury. Boron might be beneficial as a supplement in MI and may be a good candidate substance for anti-fibrosis approach.
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http://dx.doi.org/10.1038/s41598-020-73864-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553911PMC
October 2020

Response to 'Metabolism reprogramming: new insights of Dlk1 into cardiac fibrosis'.

Authors:
Djamel Lebeche

Eur Heart J 2019 11;40(43):3575

Cardiovascular Research Center, Department of Medicine, Icahn School of Medicine at Mount Sinai, 1 Gustave Levy Place, New York, NY, USA.

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http://dx.doi.org/10.1093/eurheartj/ehz604DOI Listing
November 2019

A role for calcium in resistin transcriptional activation in diabetic hearts.

Sci Rep 2018 10 23;8(1):15633. Epub 2018 Oct 23.

Cardiovascular Research Institute, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, 10029, USA.

The adipokine resistin has been proposed to link obesity, insulin resistance and diabetes. We have previously reported that diabetic hearts express high levels of resistin while overexpression of resistin in adult rat hearts gives rise to a phenotype resembling diabetic cardiomyopathy. The transcriptional regulation of resistin in diabetic cardiac tissue is currently unknown. This study investigated the mechanism of resistin upregulation and the role of Serca2a in its transcriptional suppression. We demonstrate that restoration of Ca homeostasis in diabetic hearts, through normalization of Serca2a function genetically and pharmacologically, suppressed resistin expression via inhibition of NFATc. H9c2 myocytes stimulated with high-glucose concentration or Ca time-dependently increased NFATc and resistin expression while addition of the Ca chelator BAPTA-AM attenuated this effect. NFATc expression was enhanced in hearts from ob/ob diabetic and from cardiac-specific Serca2a mice. Similarly, NFATc increased resistin expression in myocytes cultured in low glucose while the NFATc inhibitor VIVIT blocked glucose-induced resistin expression, suggesting that hyperglycemia/diabetes induces resistin expression possibly through NFATc activation. Interestingly, overexpression of Serca2a or VIVIT mitigated glucose-stimulated resistin and NFATc expression and enhanced AMPK activity, a downstream target of resistin signaling. NFATc direct activation of resistin was verified by resistin promoter luciferase activity and chromatin-immunoprecipitation analysis. Interestingly, activation of Serca2a by a novel agonist, CDN1163, mirrored the effects of AAV9-Serca2a gene transfer on resistin expression and its promoter activity and AMPK signaling in diabetic mice. These findings parse a role for Ca in resistin transactivation and provide support that manipulation of Serca2a-NFATc-Resistin axis might be useful in hyper-resistinemic conditions.
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http://dx.doi.org/10.1038/s41598-018-34112-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199245PMC
October 2018

FTO-Dependent N-Methyladenosine Regulates Cardiac Function During Remodeling and Repair.

Circulation 2019 01;139(4):518-532

Cardiovascular Research Center, Icahn School of Medicine, Mount Sinai, NY.

Background: Despite its functional importance in various fundamental bioprocesses, studies of N-methyladenosine (m6A) in the heart are lacking. Here, we show that the FTO (fat mass and obesity-associated protein), an m6A demethylase, plays a critical role in cardiac contractile function during homeostasis, remodeling, and regeneration.

Methods: We used clinical human samples, preclinical pig and mouse models, and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO by using adeno-associated virus serotype 9 (in vivo), adenovirus (both in vivo and in vitro), and small interfering RNAs (in vitro) to study its function in regulating cardiomyocyte m6A, calcium dynamics and contractility, and cardiac function postischemia. We performed methylated (m6A) RNA immunoprecipitation sequencing to map transcriptome-wide m6A, and methylated (m6A) RNA immunoprecipitation quantitative polymerase chain reaction assays to map and validate m6A in individual transcripts, in healthy and failing hearts, and in myocytes.

Results: We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is performed by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. In addition, we demonstrate that FTO overexpression in mouse models of myocardial infarction decreased fibrosis and enhanced angiogenesis.

Conclusions: Collectively, our study demonstrates the functional importance of the FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.118.033794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400591PMC
January 2019

Deletion of delta-like 1 homologue accelerates fibroblast-myofibroblast differentiation and induces myocardial fibrosis.

Eur Heart J 2019 03;40(12):967-978

Department of Medicine, Cardiovascular Research Institute.

Aims: Myocardial fibrosis is associated with profound changes in ventricular architecture and geometry, resulting in diminished cardiac function. There is currently no information on the role of the delta-like homologue 1 (Dlk1) in the regulation of the fibrotic response. Here, we investigated whether Dlk1 is involved in cardiac fibroblast-to-myofibroblast differentiation and regulates myocardial fibrosis and explored the molecular mechanism underpinning its effects in this process.

Methods And Results: Using Dlk1-knockout mice and adenoviral gene delivery, we demonstrate that overexpression of Dlk1 in cardio-fibroblasts resulted in inhibition of fibroblast proliferation and differentiation into myofibroblasts. This process is mediated by TGF-β1 signalling, since isolated fibroblasts lacking Dlk1 exhibited a higher activation of the TGF-β1/Smad-3 pathway at baseline, leading to an earlier acquisition of a myofibroblast phenotype. Likewise, Dlk1-null mice displayed increased TGF-β1/Smad3 cardiac activity, resulting in infiltration/accumulation of myofibroblasts, induction and deposition of extra-domain A-fibronectin isoform and collagen, and activation of pro-fibrotic markers. Furthermore, these profibrotic events were associated with disrupted myofibril integrity, myocyte hypertrophy, and cardiac dysfunction. Interestingly, Dlk1 expression was down-regulated in ischaemic human and porcine heart tissues. Mechanistically, miR-370 mediated Dlk1's regulation of cardiac fibroblast-myofibroblast differentiation by directly targeting TGFβ-R2/Smad-3 signalling, while the Dlk1 canonical target, Notch pathway, does not seem to play a role in this process.

Conclusion: These findings are the first to demonstrate an inhibitory role of Dlk1 of cardiac fibroblast-to-myofibroblast differentiation by interfering with TGFβ/Smad-3 signalling in the myocardium. Given the deleterious effects of continuous activation of this pathway, we propose Dlk1 as a new potential candidate for therapy in cases where aberrant TGFβ signalling leads to chronic fibrosis.
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http://dx.doi.org/10.1093/eurheartj/ehy188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6427089PMC
March 2019

Molecular Imaging of Apoptosis in Ischemia Reperfusion Injury With Radiolabeled Duramycin Targeting Phosphatidylethanolamine: Effective Target Uptake and Reduced Nontarget Organ Radiation Burden.

JACC Cardiovasc Imaging 2018 12 14;11(12):1823-1833. Epub 2018 Feb 14.

Icahn School of Medicine at Mount Sinai, New York, New York. Electronic address:

Objectives: The purpose of this study was to evaluate the feasibility of imaging apoptosis in experimental ischemia-reperfusion model by technetium-99m (Tc)-labeled Duramycin, and compare it to an established tracer, Tc-labeled Annexin-V, which has a relative disadvantage of high radiation burden to nontarget organs.

Background: During apoptosis, the cell membrane phospholipids-phosphatidylserine (PS) and phosphatidylethanolamine (PE) are exposed and can be targeted by Annexin-V and Duramycin, respectively, for in vivo imaging. Identification of a reversible cell death process should permit therapeutic intervention to help reduce myocyte loss and left ventricle dysfunction.

Methods: In a 40-min left coronary artery ischemia-reperfusion model in 17 rabbits, 7 mCi of Tc-labeled Duramycin (n = 10), Tc-linear Duramycin (a negative tracer control; n = 3), or Tc-Annexin-V (a positive tracer-control; n = 4) were intravenously administered 30 min after reperfusion. Of the 10 Duramycin group animals, 4 animals were treated with an antiapoptotic agent, minocycline at the time of reperfusion. In vivo and ex vivo micro-single-photon emission computed tomography (μSPECT) and micro-computed tomography (μCT) imaging was performed 3 h after reperfusion, followed by quantitative assessment of tracer uptake and pathological characterization. Fluorescent Duramycin and Annexin-V were injected in 4 rats to visualize colocalization in infarct areas in a 40-min left coronary artery occlusion and 30-min reperfusion model.

Results: Intense uptake of Duramycin and Annexin-V was observed in the apical (infarcted) areas. The percent injected dose per gram uptake of Duramycin in apical region (0.751 ± 0.262%) was significantly higher than remote area in same animals (0.045 ± 0.029%; p < 0.01). Duramycin uptake was insignificantly lower than Annexin-V uptake (1.23 ± 0.304%; p > 0.01) but demonstrated substantially lower radiation burden to kidneys (0.358 ± 0.210% vs. 1.58 ± 0.316%, respectively; p < 0.001). Fluorescence studies with Duramycin and Annexin V showed colocalization in infarct areas. Minocycline treatment substantially resolved Duramycin uptake (0.354% ± 0.0624%; p < 0.01).

Conclusions: Duramycin is similarly effective in imaging apoptotic cell death as Annexin-V with lower nontarget organ radiation. Clinical feasibility of apoptosis imaging with a PE-seeking tracer should be tested.
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http://dx.doi.org/10.1016/j.jcmg.2017.11.037DOI Listing
December 2018

HAX-1 regulates SERCA2a oxidation and degradation.

J Mol Cell Cardiol 2018 01 21;114:220-233. Epub 2017 Nov 21.

Department of Pharmacology and Systems Physiology, University of Cincinnati, College of Medicine, Cincinnati, OH, USA; Molecular Biology Division, Biomedical Research Foundation, Academy of Athens, Athens, Greece. Electronic address:

Ischemia/reperfusion injury is associated with contractile dysfunction and increased cardiomyocyte death. Overexpression of the hematopoietic lineage substrate-1-associated protein X-1 (HAX-1) has been shown to protect from cellular injury but the function of endogenous HAX-1 remains obscure due to early lethality of the knockout mouse. Herein we generated a cardiac-specific and inducible HAX-1 deficient model, which uncovered an unexpected role of HAX-1 in regulation of sarco/endoplasmic reticulum Ca-ATPase (SERCA2a) in ischemia/reperfusion injury. Although ablation of HAX-1 in the adult heart elicited no morphological alterations under non-stress conditions, it diminished contractile recovery and increased infarct size upon ischemia/reperfusion injury. These detrimental effects were associated with increased loss of SERCA2a. Enhanced SERCA2a degradation was not due to alterations in calpain and calpastatin levels or calpain activity. Conversely, HAX-1 overexpression improved contractile recovery and maintained SERCA2a levels. The regulatory effects of HAX-1 on SERCA2a degradation were observed at multiple levels, including intact hearts, isolated cardiomyocytes and sarcoplasmic reticulum microsomes. Mechanistically, HAX-1 ablation elicited increased production of reactive oxygen species at the sarco/endoplasic reticulum compartment, resulting in SERCA2a oxidation and a predisposition to its proteolysis. This effect may be mediated by NAPDH oxidase 4 (NOX4), a novel binding partner of HAX-1. Accordingly, NOX inhibition with apocynin abrogated the effects of HAX-1 ablation in hearts subjected to ischemia/reperfusion injury. Taken together, our findings reveal a role of HAX-1 in the regulation of oxidative stress and SERCA2a degradation, implicating its importance in calcium homeostasis and cell survival pathways.
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http://dx.doi.org/10.1016/j.yjmcc.2017.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5801168PMC
January 2018

SERCA control of cell death and survival.

Cell Calcium 2018 01 12;69:46-61. Epub 2017 Jul 12.

Cardiovascular Research Institute, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA; Diabetes, Obesity and Metabolism Institute, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA; Graduate School of Biological Sciences, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, USA. Electronic address:

Intracellular calcium (Ca is a critical coordinator of various aspects of cellular physiology. It is increasingly apparent that changes in cellular Ca dynamics contribute to the regulation of normal and pathological signal transduction that controls cell growth and survival. Aberrant perturbations in Ca homeostasis have been implicated in a range of pathological conditions, such as cardiovascular diseases, diabetes, tumorigenesis and steatosis hepatitis. Intracellular Ca concentrations are therefore tightly regulated by a number of Ca handling enzymes, proteins, channels and transporters located in the plasma membrane and in Ca storage organelles, which work in concert to fine tune a temporally and spatially precise Ca signal. Chief amongst them is the sarco/endoplasmic reticulum (SR/ER) Ca ATPase pump (SERCA) which actively re-accumulates released Ca back into the SR/ER, therefore maintaining Ca homeostasis. There are at least 14 different SERCA isoforms encoded by three ATP2A1-3 genes whose expressions are species- and tissue-specific. Altered SERCA expression and activity results in cellular malignancy and induction of ER stress and ER stress-associated apoptosis. The role of SERCA misregulation in the control of apoptosis in various cell types and disease setting with prospective therapeutic implications is the focus of this review. Ca is a double edge sword for both life as well as death, and current experimental evidence supports a model in which Ca homeostasis and SERCA activity represent a nodal point that controls cell survival. Pharmacological or genetic targeting of this axis constitutes an incredible therapeutic potential to treat different diseases sharing similar biological disorders.
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http://dx.doi.org/10.1016/j.ceca.2017.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748262PMC
January 2018

Empagliflozin Improves Left Ventricular Diastolic Dysfunction in a Genetic Model of Type 2 Diabetes.

Cardiovasc Drugs Ther 2017 Jun;31(3):233-246

Cardiovascular Research Institute, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.

Purpose: Cardiovascular (CV) diseases in type 2 diabetes (T2DM) represent an enormous burden with high mortality and morbidity. Sodium-glucose cotransporter 2 (SGLT2) inhibitors have recently emerged as a new antidiabetic class that improves glucose control, as well as body weight and blood pressure with no increased risk of hypoglycemia. The first CV outcome study terminated with empagliflozin, a specific SGLT2 inhibitor, has shown a reduction in CV mortality and in heart failure hospitalization, suggesting a beneficial impact on cardiac function which remains to be demonstrated. This study was designed to examine the chronic effect of empagliflozin on left ventricular (LV) systolic and diastolic functions in a genetic model of T2DM, ob/ob mice.

Methods And Results: Cardiac phenotype was characterized by echocardiography, in vivo hemodynamics, histology, and molecular profiling. Our results demonstrate that empagliflozin significantly lowered HbA1c and slightly reduced body weight compared to vehicle treatment with no obvious changes in insulin levels. Empagliflozin also improved LV maximum pressure and in vivo indices of diastolic function. While systolic function was grossly not affected in both groups at steady state, response to dobutamine stimulation was significantly improved in the empagliflozin-treated group, suggesting amelioration of contractile reserve. This was paralleled by an increase in phospholamban (PLN) phosphorylation and increased SERCA2a/PLN ratio, indicative of enhanced SERCA2a function, further supporting improved cardiac relaxation and diastolic function. In addition, empagliflozin reconciled diabetes-associated increase in MAPKs and dysregulated phosphorylation of IRS1 and Akt, leading to improvement in myocardial insulin sensitivity and glucose utilization.

Conclusion: The data show that chronic treatment with empagliflozin improves diastolic function, preserves calcium handling and growth signaling pathways and attenuates myocardial insulin resistance in ob/ob mice, findings suggestive of a potential clinical utility for empagliflozin in the treatment of diastolic dysfunction.
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http://dx.doi.org/10.1007/s10557-017-6734-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681671PMC
June 2017

The Probability of Inconstancy in Assessment of Cardiac Function Post-Myocardial Infarction in Mice.

Cardiovasc Pharm Open Access 2016 9;5(5). Epub 2016 Sep 9.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, USA.

In the present study, we explore the inherent variability that leads to overlaps in cardiac functional parameters between control and post-myocardial infarction (MI) mice. Heart failure was induced by Left Coronary Artery (LCA) ligation in mice. Average Ejection Fraction (EF) measured by echocardiography was lower in MI mice compared to control, but exhibited higher Standard Deviation (SD) and Standard Error (SEM), notably in 2D mode. Fractional Shortening (FS) showed a higher degree of overlap between MI and control mice even though the mean values were significantly different. Hemodynamic measurements of EF resulted in greater SD, SEM, ± 95% confidence intervals, and effect size. In comparing echocardiography at different time points, EF and FS were consistent by mean, but had apparent fluctuation in individual tracks, which were more obvious in MI than control mice. Hemodynamic measurements showed more complexity in data collection in mice . MI size showed variability that correlated with severity of cardiac function. These studies show that there is inherent variability in functional cardiac parameters after induction of heart failure by MI in mice. Analysis of these parameters by traditional statistical methods is insufficient, and we propose a more robust statistical analysis for proper data interpretation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5130155PMC
http://dx.doi.org/10.4172/2329-6607.1000195DOI Listing
September 2016

SERCA2 Deficiency Impairs Pancreatic β-Cell Function in Response to Diet-Induced Obesity.

Diabetes 2016 10 3;65(10):3039-52. Epub 2016 Aug 3.

Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, IN Department of Medicine, Indiana University School of Medicine, Indianapolis, IN Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN Roudebush VA Medical Center, Indianapolis, IN

The sarcoendoplasmic reticulum (ER) Ca(2+) ATPase 2 (SERCA2) pump is a P-type ATPase tasked with the maintenance of ER Ca(2+) stores. Whereas β-cell SERCA2 expression is reduced in diabetes, the role of SERCA2 in the regulation of whole-body glucose homeostasis has remained uncharacterized. To this end, SERCA2 heterozygous mice (S2HET) were challenged with a high-fat diet (HFD) containing 45% of kilocalories from fat. After 16 weeks of the HFD, S2HET mice were hyperglycemic and glucose intolerant, but adiposity and insulin sensitivity were not different between HFD-fed S2HET mice and HFD-fed wild-type controls. Consistent with a defect in β-cell function, insulin secretion, glucose-induced cytosolic Ca(2+) mobilization, and the onset of steady-state glucose-induced Ca(2+) oscillations were impaired in HFD-fed S2HET islets. Moreover, HFD-fed S2HET mice exhibited reduced β-cell mass and proliferation, altered insulin production and proinsulin processing, and increased islet ER stress and death. In contrast, SERCA2 activation with a small molecule allosteric activator increased ER Ca(2+) storage and rescued tunicamycin-induced β-cell death. In aggregate, these data suggest a critical role for SERCA2 and the regulation of ER Ca(2+) homeostasis in the β-cell compensatory response to diet-induced obesity.
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http://dx.doi.org/10.2337/db16-0084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5033263PMC
October 2016

Small Molecular Allosteric Activator of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) Attenuates Diabetes and Metabolic Disorders.

J Biol Chem 2016 Mar 23;291(10):5185-98. Epub 2015 Dec 23.

From the Cardiovascular Research Institute and Diabetes Obesity and Metabolism Institute, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York 10029,

Dysregulation of endoplasmic reticulum (ER) Ca(2+) homeostasis triggers ER stress leading to the development of insulin resistance in obesity and diabetes. Impaired function of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) has emerged as a major contributor to ER stress. We pharmacologically activated SERCA2b in a genetic model of insulin resistance and type 2 diabetes (ob/ob mice) with a novel allosteric activator, CDN1163, which markedly lowered fasting blood glucose, improved glucose tolerance, and ameliorated hepatosteatosis but did not alter glucose levels or body weight in lean controls. Importantly, CDN1163-treated ob/ob mice maintained euglycemia comparable with that of lean mice for >6 weeks after cessation of CDN1163 administration. CDN1163-treated ob/ob mice showed a significant reduction in adipose tissue weight with no change in lean mass, assessed by magnetic resonance imaging. They also showed an increase in energy expenditure using indirect calorimetry, which was accompanied by increased expression of uncoupling protein 1 (UCP1) and UCP3 in brown adipose tissue. CDN1163 treatment significantly reduced the hepatic expression of genes involved in gluconeogenesis and lipogenesis, attenuated ER stress response and ER stress-induced apoptosis, and improved mitochondrial biogenesis, possibly through SERCA2-mediated activation of AMP-activated protein kinase pathway. The findings suggest that SERCA2b activation may hold promise as an effective therapy for type-2 diabetes and metabolic dysfunction.
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http://dx.doi.org/10.1074/jbc.M115.705012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4777852PMC
March 2016

Diabetic cardiomyopathy: is resistin a culprit?

Authors:
Djamel Lebeche

Cardiovasc Diagn Ther 2015 Oct;5(5):387-93

Cardiovascular Research Institute, Graduate School of Biological Sciences, Department of Medicine, Division of Cardiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Cardiovascular disease, including heart failure (HF), is the major cause of death in patients with diabetes. A contributing factor to the occurrence of HF in such patients is the development of diabetic cardiomyopathy. Recent evidence demonstrates that perturbations associated with adipokines secretion and signaling result in lusitropic and inotropic defects in diabetic cardiomyopathy. This perspective editorial will discuss the central role of resistin, a recently discovered adipokine, in the maladaptive cardiac phenotype seen in diabetic hearts. Given the pleiotropic effects of resistin, strategies targeting the control of resistin levels may constitute a potentially viable therapeutic utility in patients with diabetes and diabetes-induced cardiovascular diseases.
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http://dx.doi.org/10.3978/j.issn.2223-3652.2015.05.04DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4609909PMC
October 2015

Dominant negative Ras attenuates pathological ventricular remodeling in pressure overload cardiac hypertrophy.

Biochim Biophys Acta 2015 Nov 8;1853(11 Pt A):2870-84. Epub 2015 Aug 8.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA, USA.

The importance of the oncogene Ras in cardiac hypertrophy is well appreciated. The hypertrophic effects of the constitutively active mutant Ras-Val12 are revealed by clinical syndromes due to the Ras mutations and experimental studies. We examined the possible anti-hypertrophic effect of Ras inhibition in vitro using rat neonatal cardiomyocytes (NRCM) and in vivo in the setting of pressure-overload left ventricular (LV) hypertrophy (POH) in rats. Ras functions were modulated via adenovirus directed gene transfer of active mutant Ras-Val12 or dominant negative mutant N17-DN-Ras (DN-Ras). Ras-Val12 expression in vitro activates NFAT resulting in pro-hypertrophic and cardio-toxic effects on NRCM beating and Z-line organization. In contrast, the DN-Ras was antihypertrophic on NRCM, inhibited NFAT and exerted cardio-protective effects attested by preserved NRCM beating and Z line structure. Additional experiments with silencing H-Ras gene strategy corroborated the antihypertrophic effects of siRNA-H-Ras on NRCM. In vivo, with the POH model, both Ras mutants were associated with similar hypertrophy two weeks after simultaneous induction of POH and Ras-mutant gene transfer. However, LV diameters were higher and LV fractional shortening lower in the Ras-Val12 group compared to control and DN-Ras. Moreover, DN-Ras reduced the cross-sectional area of cardiomyocytes in vivo, and decreased the expression of markers of pathologic cardiac hypertrophy. In isolated adult cardiomyocytes after 2 weeks of POH and Ras-mutant gene transfer, DN-Ras improved sarcomere shortening and calcium transients compared to Ras-Val12. Overall, DN-Ras promotes a more physiological form of hypertrophy, suggesting an interesting therapeutic target for pathological cardiac hypertrophy.
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http://dx.doi.org/10.1016/j.bbamcr.2015.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4715892PMC
November 2015

Up-regulation of micro-RNA765 in human failing hearts is associated with post-transcriptional regulation of protein phosphatase inhibitor-1 and depressed contractility.

Eur J Heart Fail 2015 Aug 15;17(8):782-93. Epub 2015 Jul 15.

Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.

Aims: Impaired sarcoplasmic reticulum (SR) Ca(2+) cycling and depressed contractility, a hallmark of human and experimental heart failure, has been partially attributed to increased protein phosphatase 1 (PP-1) activity, associated with down-regulation of its endogenous inhibitor-1. The levels and activity of inhibitor-1 are reduced in failing hearts, contributing to dephosphorylation and inactivation of key calcium cycling proteins. Therefore, we investigated the mechanisms that mediate decreases in inhibitor-1 by post-transcriptional modification.

Methods And Results: Bioinformatics revealed that 17 human microRNAs may serve as modulators of inhibitor-1. However, real-time PCR analysis identified only one of these microRNAs, miR-765, as being increased in human failing hearts concomitant with decreased inhibitor-1 levels. Expression of miR-765 in HEK293 cells or mouse ventricular myocytes confirmed suppression of inhibitor-1 levels through binding of this miR-765 to the 3'-untranslated region of inhibitor-1 mRNA. To determine the functional significance of miR-765 in Ca(2+) cycling, pri-miR-765 as well as a non-translated nucleotide sequence (miR-Ctrl) were expressed in adult mouse ventricular myocytes. The inhibitor-1 expression levels were decreased, accompanied by enhanced PP-1 activity in the miR-765 cardiomyocytes, and these reflected depressed contractile mechanics and Ca(2+) transients, compared with the miR-Ctrl group. The depressive effects were associated with decreases in the phosphorylation of phospholamban and SR Ca(2+) load. These miR-765 negative inotropic effects were abrogated in inhibitor-1-deficient cardiomyocytes, suggesting its apparent specificity for inhibitor-1.

Conclusions: miR-765 levels are increased in human failing hearts. Such increases may contribute to depressed cardiac function through reduced inhibitor-1 expression and enhanced PP-1 activity, associated with reduced SR Ca(2+) load.
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http://dx.doi.org/10.1002/ejhf.323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5693221PMC
August 2015

RAF1 mutations in childhood-onset dilated cardiomyopathy.

Nat Genet 2014 Jun 28;46(6):635-639. Epub 2014 Apr 28.

Department of Pediatrics, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, USA.

Dilated cardiomyopathy (DCM) is a highly heterogeneous trait with sarcomeric gene mutations predominating. The cause of a substantial percentage of DCMs remains unknown, and no gene-specific therapy is available. On the basis of resequencing of 513 DCM cases and 1,150 matched controls from various cohorts of distinct ancestry, we discovered rare, functional RAF1 mutations in 3 of the cohorts (South Indian, North Indian and Japanese). The prevalence of RAF1 mutations was ~9% in childhood-onset DCM cases in these three cohorts. Biochemical studies showed that DCM-associated RAF1 mutants had altered kinase activity, resulting in largely unaltered ERK activation but in AKT that was hyperactivated in a BRAF-dependent manner. Constitutive expression of these mutants in zebrafish embryos resulted in a heart failure phenotype with AKT hyperactivation that was rescued by treatment with rapamycin. These findings provide new mechanistic insights and potential therapeutic targets for RAF1-associated DCM and further expand the clinical spectrum of RAF1-related human disorders.
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http://dx.doi.org/10.1038/ng.2963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4049514PMC
June 2014

Multifaceted roles of miR-1s in repressing the fetal gene program in the heart.

Cell Res 2014 Mar 31;24(3):278-92. Epub 2014 Jan 31.

Mindich Child Health and Development Institute, Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1040, New York, NY 10029, USA.

miRNAs are an important class of regulators that play roles in cellular homeostasis and disease. Muscle-specific miRNAs, miR-1-1 and miR-1-2, have been found to play important roles in regulating cell proliferation and cardiac function. Redundancy between miR-1-1 and miR-1-2 has previously impeded a full understanding of their roles in vivo. To determine how miR-1s regulate cardiac function in vivo, we generated mice lacking miR-1-1 and miR-1-2 without affecting nearby genes. miR-1 double knockout (miR-1 dKO) mice were viable and not significantly different from wild-type controls at postnatal day 2.5. Thereafter, all miR-1 dKO mice developed dilated cardiomyopathy (DCM) and died before P17. Massively parallel sequencing showed that a large portion of upregulated genes after deletion of miR-1s is associated with the cardiac fetal gene program including cell proliferation, glycolysis, glycogenesis, and fetal sarcomere-associated genes. Consistent with gene profiling, glycogen content and glycolytic rates were significantly increased in miR-1 dKO mice. Estrogen-related Receptor β (Errβ) was identified as a direct target of miR-1, which can regulate glycolysis, glycogenesis, and the expression of sarcomeric proteins. Cardiac-specific overexpression of Errβ led to glycogen storage, cardiac dilation, and sudden cardiac death around 3-4 weeks of age. We conclude that miR-1 and its primary target Errβ act together to regulate the transition from prenatal to neonatal stages by repressing the cardiac fetal gene program. Loss of this regulation leads to a neonatal DCM.
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http://dx.doi.org/10.1038/cr.2014.12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3945888PMC
March 2014

Differential patterns of replacement and reactive fibrosis in pressure and volume overload are related to the propensity for ischaemia and involve resistin.

J Physiol 2013 Nov 9;591(21):5337-55. Epub 2013 Sep 9.

D. Lebeche: Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1030, New York, NY 10029, USA.

Pathological left ventricle (LV) hypertrophy (LVH) results in reactive and replacement fibrosis. Volume overload LVH (VOH) is less profibrotic than pressure overload LVH (POH). Studies attribute subendocardial fibrosis in POH to ischaemia, and reduced fibrosis in VOH to collagen degradation favouring dilatation. However, the mechanical origin of the relative lack of fibrosis in VOH is incompletely understood. We hypothesized that reduced ischaemia propensity in VOH compared to POH accounted for the reduced replacement fibrosis, along with reduced reactive fibrosis. Rats with POH (ascending aortic banding) evolved into either compensated-concentric POH (POH-CLVH) or dilated cardiomyopathy (POH-DCM); they were compared to VOH (aorta-caval fistula). We quantified LV fibrosis, structural and haemodynamic factors of ischaemia propensity, and the activation of profibrotic pathways. Fibrosis in POH-DCM was severe, subendocardial and subepicardial, in contrast with subendocardial fibrosis in POH-CLVH and nearly no fibrosis in VOH. The propensity for ischaemia was more important in POH versus VOH, explaining different patterns of replacement fibrosis. LV collagen synthesis and maturation, and matrix metalloproteinase-2 expression, were more important in POH. The angiotensin II-transforming growth-factor β axis was enhanced in POH, and connective tissue growth factor (CTGF) was overexpressed in all types of LVH. LV resistin expression was markedly elevated in POH, mildly elevated in VOH and independently reflected chronic ischaemic injury after myocardial infarction. In vitro, resistin is induced by angiotensin II and induces CTGF in cardiomyocytes. Based on these findings, we conclude that a reduced ischaemia propensity and attenuated upstream reactive fibrotic pathways account for the attenuated fibrosis in VOH versus POH.
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http://dx.doi.org/10.1113/jphysiol.2013.258731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3936371PMC
November 2013

Therapeutic cardiac-targeted delivery of miR-1 reverses pressure overload-induced cardiac hypertrophy and attenuates pathological remodeling.

J Am Heart Assoc 2013 Apr 23;2(2):e000078. Epub 2013 Apr 23.

Cardiovascular Research Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Background: MicroRNAs (miRNAs) play a key role in the development of heart failure, and recent studies have shown that the muscle-specific miR-1 is a key regulator of cardiac hypertrophy. We tested the hypothesis that chronic restoration of miR-1 gene expression in vivo will regress hypertrophy and protect against adverse cardiac remodeling induced by pressure overload.

Methods And Results: Cardiac hypertrophy was induced by left ventricular pressure overload in male Sprague-Dawley rats subjected to ascending aortic stenosis. When the hypertrophy was established at 2 weeks after surgery, the animals were randomized to receive either an adeno-associated virus expressing miR-1 (AAV9.miR-1) or green fluorescent protein (GFP) as control (AAV9.GFP) via a single-bolus tail-vein injection. Administration of miR-1 regressed cardiac hypertrophy (left ventricular posterior wall thickness,; 2.32±0.08 versus 2.75±0.07 mm, P<0.001) and (left ventricular septum wall thickness, 2.23±0.06 versus 2.54±0.10 mm, P<0.05) and halted the disease progression compared with control-treated animals, as assessed by echocardiography (fractional shortening, 37.60±5.01% versus 70.68±2.93%, P<0.05) and hemodynamic analyses (end-systolic pressure volume relationship/effective arterial elastance, 1.87±0.46 versus 0.96±0.38, P<0.05) after 7 weeks of treatment. Additionally, miR-1 replacement therapy lead to a marked reduction of myocardial fibrosis, an improvement in calcium handling, inhibition of apoptosis, and inactivation of the mitogen-activated protein kinase signaling pathways, suggesting a favorable effect on preventing the maladaptive ventricular remodeling. We also identified and validated a novel bona fide target of miR-1, Fibullin-2 (Fbln2), a secreted protein implicated in extracellular matrix remodeling.

Conclusions: Taken together, our findings suggest that restoration of miR-1 gene expression is a potential novel therapeutic strategy to reverse pressure-induced cardiac hypertrophy and prevent maladaptive cardiac remodeling.
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http://dx.doi.org/10.1161/JAHA.113.000078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3647279PMC
April 2013

Bone morphogenetic protein-2 decreases microRNA-30b and microRNA-30c to promote vascular smooth muscle cell calcification.

J Am Heart Assoc 2012 Dec 19;1(6):e003905. Epub 2012 Dec 19.

Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.

Background: Vascular calcification resembles bone formation and involves vascular smooth muscle cell (SMC) transition to an osteoblast-like phenotype to express Runx2, a master osteoblast transcription factor. One possible mechanism by which Runx2 protein expression is induced is downregulation of inhibitory microRNAs (miR).

Methods And Results: Human coronary artery SMCs (CASMCs) treated with bone morphogenetic protein-2 (BMP-2; 100 ng/mL) demonstrated a 1.7-fold (P<0.02) increase in Runx2 protein expression at 24 hours. A miR microarray and target prediction database analysis independently identified miR-30b and miR-30c (miR-30b-c) as miRs that regulate Runx2 expression. Real-time-polymerase chain reaction confirmed that BMP-2 decreased miR-30b and miR-30c expression. A luciferase reporter assay verified that both miR-30b and miR-30c bind to the 3'-untranslated region of Runx2 mRNA to regulate its expression. CASMCs transfected with antagomirs to downregulate miR-30b-c demonstrated significantly increased Runx2, intracellular calcium deposition, and mineralization. Conversely, forced expression of miR-30b-c by transfection with pre-miR-30b-c prevented the increase in Runx2 expression and mineralization of SMCs. Calcified human coronary arteries demonstrated higher levels of BMP-2 and lower levels of miR-30b than did noncalcified donor coronary arteries.

Conclusions: BMP-2 downregulates miR-30b and miR-30c to increase Runx2 expression in CASMCs and promote mineralization. Strategies that modulate expression of miR-30b and miR-30c may influence vascular calcification.
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http://dx.doi.org/10.1161/JAHA.112.003905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3540659PMC
December 2012

Na+/Ca2+ exchanger-1 protects against systolic failure in the Akitains2 model of diabetic cardiomyopathy via a CXCR4/NF-κB pathway.

Am J Physiol Heart Circ Physiol 2012 Aug 18;303(3):H353-67. Epub 2012 May 18.

Cardiovascular Research Institute, Mount Sinai School of Medicine, New York, New York 10029, USA.

Diabetic cardiomyopathy is characterized, in part, by calcium handling imbalances associated with ventricular dysfunction. The cardiac Na(+)/Ca(2+) exchanger 1 (NCX1) has been implicated as a compensatory mechanism in response to reduced contractility in the heart; however, its role in diabetic cardiomyopathy remains unknown. We aimed to fully characterize the Akita(ins2) murine model of type 1 diabetes through assessing cardiac function and NCX1 regulation. The CXCL12/CXCR4 chemokine axis is well described in its cardioprotective effects via progenitor cell recruitment postacute myocardial infarction; however, it also functions in regulating calcium dependent processes in the cardiac myocyte. We therefore investigated the potential impact of CXCR4 in diabetic cardiomyopathy. Cardiac performance in the Akita(ins2) mouse was monitored using echocardiography and in vivo pressure-volume analysis. The Akita(ins2) mouse is protected against ventricular systolic failure evident at both 5 and 12 mo of age. However, the preserved contractility was associated with a decreased sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a)/phospholamban ratio and increased NCX1 content. Direct myocardial injection of adenovirus encoding anti-sense NCX1 significantly decreased NCX1 expression and induced systolic failure in the Akita(ins2) mouse. CXCL12 and CXCR4 were both upregulated in the Akita(ins2) heart, along with an increase in IκB-α and NF-κB p65 phosphorylation. We demonstrated that CXCR4 activation upregulates NCX1 expression through a NF-κB-dependent signaling pathway in the cardiac myocyte. In conclusion, the Akita(ins2) type 1 diabetic model is protected against systolic failure due to increased NCX1 expression. In addition, our studies reveal a novel role of CXCR4 in the diabetic heart by regulating NCX1 expression via a NF-κB-dependent mechanism.
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http://dx.doi.org/10.1152/ajpheart.01198.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3423163PMC
August 2012
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